The present invention generally relates to compounds, methods and compositions useful in identifying a cell phenotype, and more specifically to compounds, methods and compositions useful in differentiating between a cell having a malignant phenotype, a cell having a pre-malignant phenotype and a cell having a non-malignant phenotype, or a metabolically impaired cell and a metabolically normal cell or a maternal versus a fetal cell. More particularly the invention relates to differential staining of cells in a biological sample, cytological or histological preparation. The present invention further relates to use of the compounds, methods and compositions for drug discovery, target validation, diagnosis, drug therapy monitoring and monitoring of disease progress.
For oncological diagnosis it is necessary to determine the presence of tumor cells, malignant or pre-malignant, among other normal or reactive cells in a tested specimen, and if possible, to determine the type of tumor and the organ from which it was derived.
The assessment of pathologically relevant traits in living cells is commonly performed by means of interpretation of cellular morphology or by testing of functional aspects such as motility [Alberts et al., Molecular Biology of the Cell. 3rd ed. New York and London (1994)], vitality, anchorage independent growth [Hamburger and Salmon, Science (1976) 197:461-463] and others. By contrast, fixed cells and tissue samples render themselves to histological assessment. For example, malignancy or cancer give rise to morphological changes that give rise to phenotypic changes. Traditionally, these assessments are performed by means of light microscopy following staining of the specimen with a dye or a combination of dyes in order to make the cells visible to the human eye. Thereafter, oncodiagnosis is established based on morphological features, such as, tissue organization, structure of nuclei and cytoplasm, nuclei to cytoplasm ratio, etc. as suggested by Boyd [Boyd, “A textbook of Pathology” Philadelphia: Lea & Febiger, 8th edition (1970)] “the chief characteristics of the neoplastic cell, as revealed by the light microscope, are nuclear and chromosomal aberrations, decreased cytoplasmic-nuclear ratio, a coarse irregular chromatin network, and larger nucleoli than normal”. In addition, metabolic disorders are characterized by the presence of metabolically impaired cells having an altered (impaired) phenotype as compared with the phenotype of a metabolically normal cell.
Yet, cytological assessments do not allow precise identification of cancer cells among the normal cellular population in a given preparation. One reason is that, in some cases, non-malignant cells mimic morphologic characteristics of cancer cells. For example, reactive mesothelium cells, present in serous effusions, often display atypical features such as multinucleation, nuclear hyperchromatism, eccentric position of the nucleus and cytoplasmic basophilia, features which are typically characteristic of cancer cells [Brownlow et al., Equine Vet. J. (1982) 14(1):86-88; Hansen et al., Am. J. Med. (1984) 77:887; Petrova et al., Lab. Delo. (1989) 6:9-11; Bibbo et al. (1976A) in Wied et al., “Compendium on Diagnostic Cytology” Chicago: Tutorial of Cytology, 7th Edition (1976)]. In sputum cytology, for example, vacuolated cells could indicate cancer, but also reactive glandular cells, in particular in patients with pneumonia [Bibbo (1976B) in Wied et al., “Compendium on Diagnostic Cytology” Chicago: Tutorial of Cytology, 7th Edition (1976)]. In addition, some malignant cells appear to have normal morphology therefore oncocytology suffers from false negative and false positive diagnoses [Morell et al., Obstet. Gynecology. (1982) 60:41-45].
Thus, despite the long list of classical cytological methods, the efficiency of cytological diagnosis is largely determined by the qualification and experience of the cytopathologist, and by his abilities to analyze the obtained data.
The classical cytological techniques are in many cases supplemented by a group of technologies, which are based on compounds of high affinity and specificity, known as immunohistochemistry, immunocytochemistry and in situ hybridization. These technologies are used for phenotypic characterization of tumors (e.g., by implication of antibodies to intermediate filaments), detection of tumor markers of prognostic value (e.g., detection of p53 antigen), as well as the detection of other oncogenic expression features and nucleic acid sequences. Despite the high specificity of these techniques to detect malignancies, none of these techniques provides a pan-malignant tool for oncodiagnosis.
The three most common types of clinical specimens used in modern diagnostic cytology are exfoliated cytology specimens, fine needle aspiration biopsy (FNAB) specimens, and cervical vaginal smears.
Exfoliative cytology deals with the phenomenon of exfoliation of cancer cells from the surface of tumors early in the progression of the disease, even in the pre-invasive stages thereof [Boyd, “A textbook of Pathology” Philadelphia: Lea & Febiger, 8th edition (1970)]. The exfoliated cells can be found in numerous body fluids such as pleural, peritoneal, pericardial, cerebrospinal, ocular, joint, gastrointestinal fluids, as well as in respiratory specimens, skin and mucosal samples and urine. These exfoliated cells can be collected by means of brushings, washings, direct puncture, drainage, touch preparations, scrapings, etc.
Fine needle aspiration (FNA) biopsy is a form of diagnostic biopsy that uses thin (fine) needles (22-gauge or narrower) to obtain cellular samples from within the body using a minimal invasive mode of body penetration. Fine needle aspirated cellular samples are then examined microscopically to detect the presence of various disease processes [NMLLS, “Fine Needle Aspiration Biopsy (FNAB) Techniques; Approved Guideline”, NMLLS document GP20-A (1996)]. As opposed to exfoliate cytology, FNAB is directed towards the analysis of lesions or masses and is therefore regarded as a more direct diagnostic approach. FNAB typically encompasses lesions and masses from the breast, thyroid glands, head, neck, prostate, chest, lungs, abdomen, etc.
Cervical vaginal smears are scrapings from the female reproductive tract aimed at enabling the diagnosis of numerous types of atypia, some of neoplastic nature, others of pre-malignant nature, or other pathological processes.
All types of cellular samples must be transferred to a slide, processed and stained in order to enable visualization by means of light microscopy.
At present, diagnostic cytology is typically based on a rather narrow set of staining methods and compositions, some of which appeared many years ago, but are still being extensively used in present clinical cytology [Kjeldsberg and Kniger, Body fluids, Chicago (1987); Koss, Diagnostic cytology and its histopathologic basis. Third edition, Philadelphia (1979)]. This refers primarily to the alcohol-fixed Papanicolaou stain and the air-dried May-Grunwald-Giemsa (a version of which is known as Romanowsky) stain, while staining with hematoxylin and eosin, Shorr's staining for endocrine cytology and the Pappenheim method are more rarely used.
The May-Grunwald-Giemsa (MGG) staining method is widely used in clinical laboratories for the diagnosis of cells of hematopoietic origin and others. However, this method does not express tinctorial selectivity for malignant cells.
The Papanicolaou staining method was initially developed for analysis of vaginal smears [Papanicolaou and Traut, Am. Y. Obst. Gynecol. (1941) 42:193-206; Papanicolaou “Atlas of Exfoliative Cytology” Cambridge, Mass.: Harvard University Press (1954)] and allows the differentiation of cell types of stratified and simple epithelia present in alcohol-fixed smears. The Papanicolaou method (Pap smears) allows one to obtain detailed morphology of the nucleus and cytoplasm in normal and tumor cells. This approach has been also widely used in other fields of clinical cytology. It should be emphasized that the Papanicolaou staining procedure, as well as all other staining procedures, do not reveal any tinctorial selectivity for malignant cells.
Thus, none of these prior art cytological staining protocols provide selective heterochromatic or tinctorial selectivity of pre-malignant and malignant cells, metabolically impaired cells and fetal cells. There is thus a widely recognized need for, and it would be highly advantageous to have reliable and efficient cytological staining methods which detect, monitor and validate phenotypical changes in cancer cells as compared to normal cells, in metabolically impaired cells as compared to metabolically normal cells and in fetal cell as compared to maternal cell. While existing practice is reliable, it is both time consuming and in most cases not amenable to high throughput testing of cellular phenotype. Moreover, as a result of the increasing number of new genes with unknown function, as well as the need to determine the function of known genes, there is a great demand for rapid and simple methods for detecting oncogenes and tumor suppressor genes. There is a recognized need to test chemicals and drugs for mutagenicity, particularly with techniques which are readily adapted to automation for high throughput screening. Furthermore, there is a need for new type/generation of anti-cancer treatments changing the behavior/phenotype of cells from malignant to normal. Also there is a need for the correct combination of drugs in anti-cancer treatment. These requests are not fulfilled due to the lack of the screening assays and platforms.